Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system
ABSTRACT Gemcitabine, a pyrimidine nucleoside, is approved for the treatment of non-small cell lung cancer, pancreatic carcinoma, and breast cancer. Chemotherapy regimens are determined experimentally with static tissue culture systems, animal models, and in Phase I clinical trials. The aim of this study was to assess for gemcitabine-induced cell death following infusion of drug under clinically-relevant conditions of infusion rate and drug exposure in an in vitro bioreactor system.
To estimate an appropriate harvest time for cells from the bioreactor after drug treatment, we estimated the temporal relationship between gemcitabine treatment for 1 h and cell death at a later time point with monolayer growth assays (i.e., static culture). Afterward, 5.3 mg gemcitabine was infused over 0.5 h in the bioreactor, followed by mono-exponential decay, simulating patient concentration-time profiles (n = 4). Controls were run with drug-free media (n = 4). Cells were harvested from the bioreactor at a later time point and assessed for cell death by flow cytometry.
According to monolayer growth assay results, cytotoxicity became more apparent with increasing time. The E Max for cells 48 h after treatment was 50% and after 144 h, 93% (P = 0.022; t test), while flow cytometry showed complete DNA degradation by 120 h. Gemcitabine was infused in the bioreactor. The gemcitabine area under the concentration-time curve (AUC) was 56.4 microM h and the maximum concentration was 87.5 +/- 2.65 microM. Flow cytometry results were as follows: the G1 fraction decreased from 65.1 +/- 4.91 to 28.6 +/- 12% (P = 0.005) and subG1 increased from 14.1 +/- 5.28 to 42.6 +/- 9.78% (P = 0.004) relative to control. An increase in apoptotic cells was observed by TUNEL assay.
The in vitro bioreactor system will be expanded to test additional cell lines, and will serve as a useful model system for assessing the role of drug pharmacokinetics in delivery of optimized anticancer treatment.
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ABSTRACT: Recent years have shown a great deal of interest and research into the understanding of the biological and physiological roles of mechanical forces on cellular behavior. Despite these reports, in vitro screening of new molecular entities for lung ailments is still performed in static cell culture models. Failure to incorporate the effects of mechanical forces during early stages of screening could significantly reduce the success rate of drug candidates in the highly expensive clinical phases of the drug discovery pipeline. The objective of this review is to expand our current understanding of lung mechanotransduction and extend its applicability to cellular physiology and new drug screening paradigms. This review covers early in vivo studies and the importance of mechanical forces in normal lung development, use of different types of bioreactors that simulate in vivo movements in a controlled in vitro cell culture environment, and recent research using dynamic cell culture models. The cells in lungs are subjected to constant stretching (mechanical forces) in regular cycles due to involuntary expansion and contraction during respiration. The effects of stretch on normal and abnormal (disease) lung cells under pathological conditions are discussed. The potential benefits of extending dynamic cell culture models (screening in the presence of forces) and the associated challenges are also discussed in this review. Based on this review, the authors advocate the development of dynamic high throughput screening models that could facilitate the rapid translation of in vitro biology to animal models and clinical efficacy. These concepts are translatable to cardiovascular, digestive, and musculoskeletal tissues and in vitro cell systems employed routinely in drug-screening applications.Assay and Drug Development Technologies 02/2012; 10(2):137-47. DOI:10.1089/adt.2011.418 · 2.08 Impact Factor
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ABSTRACT: Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.ChemMedChem 03/2011; 6(3):457-64. DOI:10.1002/cmdc.201000447 · 3.05 Impact Factor
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ABSTRACT: The purpose of this study was to determine whether everolimus, a rapamycin derivative, might significantly enhance the cytotoxicity of gemcitabine, an antitumor drug, in two human bladder-cancer cell lines. Human bladder-cancer T24 and 5637 cells were incubated with gemcitabine and everolimus in a range of concentrations either alone or in combination for 72 h. Flow cytometry, comet assay, MTT method and optical microscopy were used to assess cell proliferation, cell cycle, DNA damage, and morphological alterations. Gemcitabine exerted an inhibitory effect on T24 and 5637 cell proliferation, in a concentration-dependent manner. Everolimus significantly reduced proliferation of 5637 bladder cancer cells (IC₃₀) at 1 μM), whereas T24 demonstrated marked resistance to everolimus treatment. A significant antiproliferative effect was obtained combining gemcitabine (100 nM) with everolimus (0.05-2 μM) with an arrest of cell cycle at S phase. Furthermore, an increase in frequency of DNA damage, apoptotic bodies, and apoptotic cells was observed when T24 and 5637 cancer cells were treated simultaneously with both drugs. Data show that in vitro combination produced a more potent antiproliferative effect when compared with single drugs.Journal of Toxicology and Environmental Health Part A 07/2012; 75(13-15):788-99. DOI:10.1080/15287394.2012.690325 · 1.83 Impact Factor